The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Characterization of Suicidal Erythrocyte Death (Eryptosis) in Dogs.

BACKGROUND/AIMS: Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. METHODS: Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. RESULTS: All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. CONCLUSION: Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.